Institute for Microbial Biotechnology & Metagenomics

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16 S rDNA primers and the unbiased assessment of thermophile diversity
(Portland Press, 2004) Baker, Gillian; Cowan, Donald A.
Our understanding of thermophile diversity is based predominantly on PCR studies of community DNA. ‘Universal’ and domain-specific rRNA gene PCR primers have historically been used for the assessment of microbial diversity without adequate regard to the degree of specificity of primer pairs to different prokaryotic groups. In a reassessment of the published primers commonly used for ‘universal’ and archaeal 16 S rDNA sequence amplification we note that substantial variations in specificity exist. An unconsidered choice of primers may therefore lead to significant bias in determination of microbial community composition. In particular, Archaea-specific primer sequences typically lack specificity for the Korarchaeota and Nanoarchaea and are often biased towards certain clades. New primer pairs specifically designed for ‘universal’ archaeal 16 S rDNA sequence amplification, with homology to all four archaeal groups, have been designed. Here we present the application of these new primers for preparation of 16 S libraries from thermophile communities.
Antarctic Dry Valley mineral soils contain unexpectedly high levels of microbial biomass
(Springer Verlag, 2002) Cowan, Donald A.; Mamais, A.; Russell, Nick A.; Sheppard, Devon M.
We have applied bioluminescent ATP detection methods to microbial enumeration in Antarctic Dry Valley mineral soils, and validated our ATP data by two independent methods. We have demonstrated that ATP measurement is a valid means of determining microbial biomass in such sites, and that the desiccated surface mineral soils of the Antarctic Dry Valleys contain cell numbers over four orders of magnitude higher than previously suggested
Biodegradation of high-concentration isopropanol by a solvent-tolerant thermophile, Bacillus pallidus
(Springer Verlag, 2002) Bustard, Mark T.; Whiting, Samantha; Cowan, Donald A.; Wright, Phillip C.
The aerobic biodegradation of high-concentration, to 24 g l –1 , 2-propanol (IPA) by a thermophilic isolate ST3, identified as Bacillus pallidus , was successfully carried out for the first time. This solvent-tolerant B. pallidus utilized IPA as the sole carbon source within a minimal salts medium. Cultivation was carried out in 100-ml shake flasks at 60°C and compared with cultivation within a 1-l stirred tank reactor (STR). Specific growth rate () was about 0.2 h–1 for both systems, with a maximum cell density of 2.4 x 10 8 cells ml–1 obtained with STR cultivation. During exponential growth and stationary phase, IPA biodegradation rates were found to be 0.14 and 0.02 g l –1h–1, respectively, in shake-flask experiments, whereas corresponding values of 0.09 and 0.018 g l –1h–1 were achievable in the STR. Generation of acetone, the major intermediate in aerobic IPA biodegradation, was also monitored as an indicator of microbial IPA utilization. Acetone levels reached a maximum of 2.2–2.3 g l–1 after 72 and 58 h for 100-ml and 1-l systems, respectively. Both IPA and acetone were completely removed from the medium following 160 and 175 h, respectively, during STR growth, although this was not demonstrated within shake-flask reactions. Growth of B. pallidus on acetone or IPA alone demonstrated that the maximum growth rate () obtainable was 0.247 h–1 at 4 g l–1 acetone and 0.202 h–1 at 8 g l–1 IPA within shake-flask cultivation. These results indicate the potential of the solvent-tolerant thermophile B. pallidus ST3 in the bioremediation of hot solvent-containing industrial waste streams.
Characterisation of campylobacter concisus strains from South Africa using amplified fragment length polymorphism (AFLP) profiling and a genomospecies-specific polymerase chain reaction (PCR) assay: Identification of novel genomospecies and correlation with clinical data
(Academic Journals, 2013) On, S.L.W.; Siemer, B. L.; Brandt, S. M.; Chung, P.; Lastovica, Albert J.
Amplified Fragment Length Polymorphism (AFLP) profiling was used to evaluate the distribution of phenotypically indistinguishable, but genetically distinct, among Campylobacter concisus strains from South Africa. A Polymerase Chain Reaction (PCR) assay described for identifying strains belonging to Genomospecies 1 and 2 was applied in this study. Forty-seven C. concisus strains were studied in total, of which 42 were of South African origin. Forty of the South African isolates were assigned to the major existing genomospecies typified by the type strain of oral origin (GS1), and reference strains from bloody diarrhoea (GS2). Eighteen South African isolates were distributed in the GS1 cluster including two oral strains. Twenty-two faecal South African isolates clustered with reference GS2 strains. Two novel genomospecies (GS 5 and 6) were inferred by their AFLP profile characteristics. Use of an existing PCR assay first described for identification of GS1 and GS2 strains generally indicated that the tool was accurate, although the novel genomospecies described here yielded an amplicon in the GS2 assay. No consistent clinical pattern among the diarrhoea South African strains could be discerned. The study extends the known genetic diversity among C. concisus, elucidates the presence of multiple genomospecies in South Africa, and confirms for the first time an association of GS1 with diarrhoea as well as the utility (with caveats) of a PCR assay for identifying GS1 and GS2 strains.
Characterisation of the arsenic resistance genes in Bacillus sp. UWC isolated from maturing fly ash acid mine drainage neutralised solids
(Academy of Science of South Africa, 2010) Musingarimi, Wicleffe; Tuffin, Marla; Cowan, Donald A.
An arsenic resistant Bacillus sp. UWC was isolated from fly ash acid mine drainage (FA-AMD) neutralised solids. A genomic library was prepared and screened in an arsenic sensitive mutant Escherichia coli strain for the presence of arsenic resistance (ars) genes. Sequence analysis of a clone conferring resistance to both sodium arsenite and sodium arsenate revealed homologues to the arsR (regulatory repressor), arsB (membrane located arsenite pump), arsC (arsenate reductase), arsD (second regulatory repressor and a metallochaperone) and arsA (ATPase) genes from known arsenic resistance operons. The Bacillus sp. UWC arsRBCDA genes were shown to be arranged in an unusual manner with the arsDA genes immediately downstream of arsC.
Cloning, expression and characterization of thermostable YdaP from Bacillus licheniformis 9A
(Acta Biochimica Polonica, 2017) Lako, Joseph D. Wani; Yengkopiong, Jada P.; Stafford, William H. L.; Tuffin, Marla; Cowan, Don A.
The Bacillus licheniformis ydaP gene encodes for a pyruvate oxidase that catalyses the oxidative decarboxylation of pyruvate to acetate and CO2. The YdaP form of this enzyme was purified about 48.6-folds to homogeneity in three steps. The enzyme was recovered in a soluble form and demonstrated significant activity on pyruvate using 2, 6-dichlorophenolindophenol (DCPIP) as an artificial electron acceptor. HPLC analysis of the YdaP-enzyme catalysed conversion of pyruvate showed acetate as the sole product, confirming the putative identity of pyruvate oxidase. Analysis of the substrate specificity showed that the YdaP enzyme demonstrated preference for short chain oxo acids; however, it was activated by 1% Triton X-100. The YdaP substrate-binding pocket from the YdaP protein differed substantially from the equivalent site in all of the so far characterized pyruvate oxidases, suggesting that the B. licheniformis YdaP might accept different substrates. This could allow more accessibility of large substrates into the active site of this enzyme. The thermostability and pH activity of the YdaP enzyme were determined, with optimums at 50ºC and pH 5.8, respectively. The amino acid residues forming the catalytic cavity were identified as Gln460 to Ala480.
A designed experiments approach to optimizing MALDI-TOF MS spectrum processing parameters enhances detection of antibiotic resistance in Campylobacter jejuni
(Frontiers Media, 2016) Penny, Christian; Lastovica, Albert J.
MALDI-TOF MS has been utilized as a reliable and rapid tool for microbial fingerprinting at the genus and species levels. Recently, there has been keen interest in using MALDI-TOF MS beyond the genus and species levels to rapidly identify antibiotic resistant strains of bacteria. The purpose of this study was to enhance strain level resolution for Campylobacter jejuni through the optimization of spectrum processing parameters using a series of designed experiments. A collection of 172 strains of C. jejuni were collected from Luxembourg, New Zealand, North America, and South Africa, consisting of four groups of antibiotic resistant isolates. The groups included: (1) 65 strains resistant to cefoperazone (2) 26 resistant to cefoperazone and beta-lactams (3) 5 strains resistant to cefoperazone, beta-lactams, and tetracycline, and (4) 76 strains resistant to cefoperazone, teicoplanin, amphotericin, B and cephalothin. Initially, a model set of 16 strains (three biological replicates and three technical replicates per isolate, yielding a total of 144 spectra) of C. jejuni was subjected to each designed experiment to enhance detection of antibiotic resistance. The most optimal parameters were applied to the larger collection of 172 isolates (two biological replicates and three technical replicates per isolate, yielding a total of 1,031 spectra). We observed an increase in antibiotic resistance detection whenever either a curve based similarity coefficient (Pearson or ranked Pearson) was applied rather than a peak based (Dice) and/or the optimized preprocessing parameters were applied. Increases in antimicrobial resistance detection were scored using the jackknife maximum similarity technique following cluster analysis. From the first four groups of antibiotic resistant isolates, the optimized preprocessing parameters increased detection respective to the aforementioned groups by: (1) 5% (2) 9% (3) 10%, and (4) 2%. An additional second categorization was created from the collection consisting of 31 strains resistant to beta-lactams and 141 strains sensitive to beta-lactams. Applying optimal preprocessing parameters, beta-lactam resistance detection was increased by 34%. These results suggest that spectrum processing parameters, which are rarely optimized or adjusted, affect the performance of MALDI-TOF MS-based detection of antibiotic resistance and can be fine-tuned to enhance screening performance.
Dissemination and survival of non-indigenous bacterial genomes in pristine Antarctic environments.
(Springer Verlag, 2005) Ah Tow, Lemese; Cowan, Donald A.
Continental Antarctic is perceived as a largely pristine environment, although certain localized regions (e.g., parts of the Ross Dependency Dry Valleys) are relatively heavy impacted by human activities. The procedures imposed on Antarctic field parties for the handling and disposal of both solid and liquid wastes are designed to minimise eutrofication and contamination (particularly by human enteric bacteria). However, little consideration has been given to the significance, if any, of less obvious forms of microbial contamination resulting from periodic human activities in Antarctica. The predominant commensal microorganism on human skin, Staphylococcus epidermidis, could be detected by PCR, in Dry Valley mineral soils collected from heavily impacted areas, but could not be detected in Dry Valley mineral soils collected from low impact and pristine areas. Cell viability of this non-enteric human commensal is rapidly lost in Dry Valley mineral soil. However, S. epidermidis can persist for long periods in Dry Valley mineral soil as non-viable cells and/or naked DNA.
Draft Genome Sequence of Gordonia lacunae BS2T
(American Society for Microbiology, 2017) Durrell, Kim; Prins, Alaric; Le Roes-Hill, Marilize
We report here the draft genome sequence of the soil bacterium Gordonia lacunae BS2T ( DSM 45085T JCM 14873T NRRL B-24551T), isolated from an estuary in Plettenberg Bay, South Africa. Analysis of the draft genome revealed that more than 40% of the secondary metabolite biosynthetic genes encode new compounds.
Effect of Fe-Mo promoters on HZSM-5 zeolitecatalyst for 1-hexene aromatization
(Elsevier B.V., 2018-11-15) Key, David; Mdleleni, Masikana
The promotional effect of Fe-Mo species introduced into HZSM-5 (Zeolyst Int.,SiO2/Al2O3 30) zeolite catalyst by the wetness impregnation method for the 1-hexene aromatiza-tion was investigated. The structure and catalytic performance for the aromatization of 1-hexeneover xFeyMo-ZSM-5 catalysts in comparison with unmodified HZSM-5 catalysts were studied.The xFeyMo-ZSM-5 catalysts contain fixed loading (5 wt%) and variable Fe/Mo ratio. The cata-lysts were characterized by BET, ICP-AES, HRSEM-EDS, HRTEM, XRD, FTIR, H2-TPR,NH3-TPD, and pyridine DRIFT spectroscopy. The characterization data confirmed the existenceof Fe and Mo species in the zeolite matrix. With Fe and Mo species implementation to HZSM-5zeolite, the amount of the acid sites decreased, but the selectivities to C9+aromatics increased.The catalyst evaluation was performed at 350°C for 6 h on-stream at atmospheric pressure usinga fixed-bed quartz tube reactor. The selectivity to products of different carbon number was affectedby the Fe/Mo ratio within the zeolite. It was found the product distribution of grouped fractions ofC1–C17+from the liquid product. The results indicate that the optimum ratio of Fe/Mo is 1–1.5.The highest selectivity for gasoline and distillate ranges was obtained for the 2.5wt%Fe2.5wt%Mo- and 3wt%Fe2wt%Mo-ZSM-5 samples, which was higher than that for parent HZSM-5 cata-lyst.
Efficient molecular cloning of environmental DNA from geothermal sediments
(Kluwer Academic Publishers, 2002) Wilkinson, Dianne E.; Jaenicke, Thomas; Cowan, Donald A.
An efficient and simple method for constructing an environmental library using mechanically sheared DNA obtained directly from geothermal sediments is presented. The method is based on blunt-end modification of DNA fragments followed by 3' -adenylation using Vent DNA polymerase and Taq DNA polymerase, respectively. The prepared DNA fragments are then ligated into a TA cloning vector and used in the transformation of Escherichia coli. This method has been successfully applied to the cloning of ORFs derived from uncultivated prokaryotes present in geothermal sediment.
Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius
(Springer Verlag, 2013) Van Zyl, L.J.; Taylor, M.P.; Eley, K.; Tuffin, Marla; Cowan, Donald A.
This study reports the expression, purification, and kinetic characterization of a pyruvate decarboxylase (PDC) from Gluconobacter oxydans . Kinetic analyses showed the enzyme to have high affinity for pyruvate (120 μM at pH 5), high catalytic efficiency (4.75×105 M−1 s−1 at pH 5), a pHopt of approximately 4.5 and an in vitro temperature optimum at approximately 55 °C. Due to in vitro thermostablity (approximately 40 % enzyme activity retained after 30 min at 65 °C), this PDC was considered to be a suitable candidate for heterologous expression in the thermophile Geobacillus thermoglucosidasius for ethanol production. Initial studies using a variety of methods failed to detect activity at any growth temperature (45–55 °C). However, the application of codon harmonization (i.e., mimicry of the heterogeneous host’s transcription and translational rhythm) yielded a protein that was fully functional in the thermophilic strain at 45 °C (as determined by enzyme activity, Western blot, mRNA detection, and ethanol productivity). Here, we describe the first successful expression of PDC in a true thermophile. Yields as high as 0.35±0.04 g/g ethanol per gram of glucose consumed were detected, highly competitive to those reported in ethanologenic thermophilic mutants. Although activities could not be detected at temperatures approaching the growth optimum for the strain, this study highlights the possibility that previously unsuccessful expression of pdcs in Geobacillus spp. may be the result of ineffective transcription/translation coupling.
High 16S rDNA bacterial diversity in glacial meltwater lake sediment, Bratina Island, Antarctica
(Springer Verlag, 2003) Sjoling, Sara; Cowan, Donald A.
The microbial diversity in maritime meltwater pond sediments from Bratina Island, Ross Sea, Antarctica was investigated by 16S rDNA-dependent molecular phylogeny. Investigations of the vertical distribution, phylogenetic composition, and spatial variability of Bacteria and Archaea in the sediment were carried out. Results revealed the presence of a highly diverse bacterial population and a significantly depthrelated composition. Assessment of 173 partial 16S rDNA clones analyzed by amplified rDNA restriction analysis (ARDRA) using tetrameric restriction enzymes (HinP1I 5'GVCGC3'and Msp I. 5'CVGG3', BioLabs) revealed 153 different bacterial OTUs (operational taxonomic units). However, only seven archaeal OTUs were detected, indicating low archaeal diversity. Based on ARDRA results, 30 bacterial clones were selected for sequencing and the sequenced clones fell into seven major lineages of the domain Bacteria; the a, c, and d subdivisions of Proteobacteria, the Cytophaga–Flavobacterium– Bacteroides, the Spirochaetaceae, and the Actinobacteria. All of the archaeal clones sequenced belonged to the group Crenarchaeota and phylogenetic analysis revealed close relationships with members of the deep-branching Group 1 Marine Archaea.
High-level diversity of tailed phages, eukaryote-associated viruses, and virophage-like elements in the metaviromes of Antarctic soils
(American Society for Microbiology, 2014) Zablocki, Olivier; van Zyl, Lonnie; Adriaenssens, Evelien M.; Rubagotti, Enrico; Tuffin, Marla; Cary, Stephen Craig; Cowan, Donald A.
The metaviromes of two distinct Antarctic hyperarid desert soil communities have been characterized. Hypolithic communities, cyanobacterium-dominated assemblages situated on the ventral surfaces of quartz pebbles embedded in the desert pavement, showed higher virus diversity than surface soils, which correlated with previous bacterial community studies. Prokaryotic viruses (i.e., phages) represented the largest viral component (particularly Mycobacterium phages) in both habitats, with an identical hierarchical sequence abundance of families of tailed phages (Siphoviridae>Myoviridae>Podoviridae). No archaeal viruses were found. Unexpectedly, cyanophages were poorly represented in both metaviromes and were phylogenetically distant from currently characterized cyanophages. Putative phage genomes were assembled and showed a high level of unaffiliated genes, mostly from hypolithic viruses. Moreover, unusual gene arrangements in which eukaryotic and prokaryotic virus-derived genes were found within identical genome segments were observed. Phycodnaviridae and Mimiviridae viruses were the second-mostabundant taxa and more numerous within open soil. Novel virophage-like sequences (within the Sputnik clade) were identified. These findings highlight high-level virus diversity and novel species discovery potential within Antarctic hyperarid soils and may serve as a starting point for future studies targeting specific viral groups.
Hypolithic and soil microbial community assembly along an aridity gradient in the Namib Desert
(Springer, 2013) Stomeo, Francesca; Valverde, Angel; Pointing, Stephen B.; McKay, Christopher P.; Warren-Rhodes, Kimberley A.; Tuffin, Marla I.; Seely, Mary; Cowan, Donald A.
The Namib Dessert is considered the oldest desert in the world and hyperarid for the last 5 million years. However, the environmental buffering provided by quartz and other translucent rocks supports extensive hypolithic microbial communities. In this study, open soil and hypolithic microbial communities have been investigated along an East–West transect characterized by an inverse fog-rainfall gradient. Multivariate analysis showed that structurally different microbial communities occur in soil and in hypolithic zones. Using variation partitioning, we found that hypolithic communities exhibited a fog-related distribution as indicated by the significant East– West clustering. Sodium content was also an important environmental factor affecting the composition of both soil and hypolithic microbial communities. Finally, although null models for patterns in microbial communities were not supported by experimental data, the amount of unexplained variation (68–97 %) suggests that stochastic processes also play a role in the assembly of such communities in the Namib Desert.
The Institute for Microbial Biotechnology and Metagenomics 2009
(UWC, 2010-01) Cowan, Donald A.
The IMBM Brochure (2009) provides a summary of the staffing, activities and outputs of the Institute for the 2009 academic year
International nonproprietary names for monoclonal antibodies: an evolving nomenclature system
(Taylor and Francis, 2022) Guimaraes Koch, Sofia S; Thorpe, Robin; Kawasaki, Nana; Lefranc, Marie-Paule
Appropriate nomenclature for all pharmaceutical substances is important for clinical development, licensing, prescribing, pharmacovigilance, and identification of counterfeits. Nonproprietary names that are unique and globally recognized for all pharmaceutical substances are assigned by the International Nonproprietary Names (INN) Programme of the World Health Organization (WHO). In 1991, the INN Programme implemented the first nomenclature scheme for monoclonal antibodies. To accompany biotechnological development, this nomenclature scheme has evolved over the years; however, since the scheme was introduced, all pharmacological substances that contained an immunoglobulin variable domain were coined with the stem -mab. To date, there are 879 INN with the stem -mab. Owing to this high number of names ending in -mab, devising new and distinguishable INN has become a challenge. The WHO INN Expert Group therefore decided to revise the system to ease this situation. The revised system was approved and adopted by the WHO at the 73rd INN Consultation held in October 2021, and the radical decision was made to discontinue the use of the well-known stem -mab in naming new antibody-based drugs and going forward, to replace it with four new stems: -tug, -bart, -mig, and -ment.
Metagenomic methods for the identification of active micro-organisms and genes in biodegradation processes.
(ASM Press, 2007) Cowan, Donald A.; Stafford, William
Metagenomics, gene discovery and the ideal biocatalyst
(Portland Press, 2004) Cowan, Donald A.; Arslanoglu, A.; Burton, Stephanie G.; Cameron, Rory A.; Baker, Gillian; Smith, Jacques J.; Meyer, Quinton
With the rapid development of powerful protein evolution and enzyme-screening technologies, there is a growing belief that optimum conditions for biotransformation processes can be established without the constraints of the properties of the biocatalyst. These technologies can then be applied to find the ‘ideal biocatalyst’ for the process. In identifying the ideal biocatalyst, the processes of gene discovery and enzyme evolution play major roles. However, in order to expand the pool genes for in vitro evolution, new technologies, which circumvent the limitations of microbial culturability, must be applied. These technologies, which currently include metagenomic library screening, gene-specific amplification methods and even full metagenomic sequencing, provide access to a volume of ‘sequence space’ that is not addressed by traditional screening.
Micro-Eukaryotic diversity in Hypolithons from Miers Valley, Antarctica
(Multidisciplinary Digital Publishing Institute (MDPI), 2013) Gokul, Jarishma K.; Valverde, Angel; Tuffin, Marla; Cary, Stephen Craig; Cowan, Donald A.
The discovery of extensive and complex hypolithic communities in both cold and hot deserts has raised many questions regarding their ecology, biodiversity and relevance in terms of regional productivity. However, most hypolithic research has focused on the bacterial elements of the community. This study represents the first investigation of micro-eukaryotic communities in all three hypolith types. Here we show that Antarctic hypoliths support extensive populations of novel uncharacterized bryophyta, fungi and protists and suggest that well known producer-decomposer-predator interactions may create the necessary conditions for hypolithic productivity in Antarctic deserts.

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